This invention relates to a method of testing for a fruit oligosaccharide. More specifically, this invention relates to a method of isolating and identifying fruit oligosaccharides in banana fruit.
Kestoses are fructosyl sucrose derivatives that are found in many plants, including bananas. In Biochem J. 48(1) 114-126 (1951), Bacon and Eldeman found bananas contain neokestose and 6-kestose, which are trisaccharides, and bifrucose, which is a tetraose. The isolation and identification of these oligosaccharides are important as they hold a potential key to the treatment of various diseases.
Fruit oligosaccharides have been found to inhibit glycolysis, which is used by various life-threatening diseases. For instance, cancer cells have a high glycolytic rate when compared to healthy tissue. By inhibiting glycolysis, fruit oligosaccharides can be used to treat cancer patients.
Fruit oligosaccharides can also act as an inhibitor in the treatment of other diseases. The inhibition of glycolytic aldolase A and C has been shown to assist in preventing neurodegenerative diseases, such as amyotropic lateral schlerosis (ALS) and Alzheimers Disease. Aldolase inhibition may also be a key to treatment of tuberculosis by stifling the metabolism of Mycobacterium tuberculosi.
Various methods of isolation and identification have been used in the past. Using an acetate conversion, fruit oligosaccharides have been analyzed using carbon-13 nuclear magnetic resonance (CNMR) to determine the isomeric kestoses in plants by Forythe et al. in Plant Physiol. 92(4) 1014-1020 (1990) and Forsythe and Feather in Carbohydr. Res. 185 315-319 (1989). 2D-NMR has also been used by Lieu et al. in Carbohydr. Res. 18; 217 43-49 (1991) to make chemical shift assignment to 6-kestose and neokestose. Matrix-assisted laser desorption/ionization coupled with time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry (ESI-MS) have been used by Sukzuki et al. in Plant Physiol. 170(8) 715-722 (2013) to confirm the presence of fructans in plant tissue, such as asparagus tissue. Other methods, such as zone electrophoresis used by Fuller et al. Biochem. J. 64(4) 657-663 (1956), paper chromatography, celite column chromatography, silica gel chromatography, and thin layer chromatography (TLC), have been used to separate and identify molecules.
Despite these advances in plant oligosaccharide isolation and identification, problems remain. Specifically, none of these methods have allowed for the isolation and structural characterization of phosphorylated or di-phosphorylated kestoses in plants. For instance, Agopia et al. n J. Agric. And Food Chem. 57(22) 10765-10771 (2009), claimed to confirm the presence of nekestose, 6-kestose, and bifurcose in banana ‘prata,’ but was unable to report any phosphorylated or di-phosphorylated kestoses.
Additionally, researchers, such as Shiomi in J. of the Faculty of Agriculture-Hokkaido University 54(4) 548-556 (1978), have used ethanol in a boiling water bath to complete extraction. Others, like Bacon and Edelman in Biochem J. 48(1) 114-126 (1951), have heated the mixtures to 80° C. for a period of 10 to 15 minutes to allow extraction. The heating of ethanol is time consuming, dangerous, destructive, and inefficient.
Thus, it is a primary objective of the invention to provide a method for isolating a fruit oligosaccharide in order to determine the oligosaccharide's disease-treatment capabilities.
Another objective of the invention is to provide a method for identifying a fruit oligosaccharide in order to determine the oligosaccharide's disease-treatment capabilities.
Yet another objective of the invention is to provide a method for characterizing phosphorylated and di-phosphorylated kestoses.
Another objective of the invention is to provide a hydride addition reaction for discernment of phosphate/sulfate ester substitution.
Yet another objective of the invention is to provide a method of extraction that does not use heat.
Another objective of the invention is to provide a method of extraction that has increased efficiency.
Yet another objective of the invention is to provide a method of extraction that is not unnecessarily degenerative.
These and other objectives, features, and advantages of the invention will become apparent from the specification and claims.